Recombinant Marinomonas sp. Translation initiation factor IF-2 (infB), partial

What is Translation Initiation Factor IF-2 and what role does it play in Marinomonas species?

Translation Initiation Factor 2 (IF-2) is a GTPase that promotes the binding of initiator fMet-tRNA to the 30S ribosomal subunit during the first phase of bacterial translation initiation. In Marinomonas species, as in other bacteria, IF-2 is essential for protein synthesis, participating in a multistep process alongside other initiation factors (IF1 and IF3) . Unlike what was previously assumed, IF-2 does not function as a tRNA carrier that delivers fMet-tRNA to the ribosome. Instead, IF-2·GTP first binds to the 30S ribosomal subunit independently, and subsequently promotes the binding of fMet-tRNA by providing anchoring interactions or inducing favorable conformational changes in the ribosome . Additionally, IF-2 plays a critical role in promoting the joining of the 50S subunit to the 30S initiation complex during the later stages of initiation .

How does Marinomonas sp. IF-2 differ from other bacterial translation initiation factors?

While specific comparative data for Marinomonas sp. IF-2 is limited, bacterial IF-2 generally differs from other translational GTPases like EF-Tu, SelB, and eukaryotic initiation factor 2 (eIF2) in its mechanism of action. Unlike these factors that form tight complexes with tRNAs (with Kd values in the nM or pM range) and deliver them as ternary complexes to the ribosome, IF-2 forms relatively weak complexes with fMet-tRNA (Kd ≈ 1 μM) that are kinetically unstable . Functionally, Marinomonas sp. IF-2 likely resembles its structural homologue eIF5B, which accelerates the joining of ribosomal subunits but has low affinity for initiator tRNA .

What is the gene structure of infB in Marinomonas species?

The infB gene encodes the translation initiation factor IF-2 in bacteria. While the complete gene structure in Marinomonas species has not been fully characterized in the provided search results, bacterial infB genes typically contain conserved domains including a G-domain responsible for GTP binding and hydrolysis, and domains involved in interactions with the ribosome and fMet-tRNA. In many bacterial species, the infB gene is part of an operon that may include other genes involved in translation or cellular processes. Further genomic analysis of different Marinomonas strains (such as the MMB-1, MMB-2, and MMB-3 strains mentioned in relation to other genetic features) would be necessary to fully characterize the specific gene structure and organization of infB in this genus .

What are the optimal methods for expressing and purifying recombinant Marinomonas sp. IF-2?

For the expression and purification of recombinant Marinomonas sp. IF-2, researchers typically employ similar approaches to those used for other bacterial IF-2 proteins, with modifications to address species-specific characteristics:

Expression System:

• E. coli BL21(DE3) or similar strains are recommended expression hosts

• Use of pET-based vectors with T7 promoter systems for high-level expression

• Addition of a His-tag (preferably at the N-terminus to avoid interference with C-terminal functions)

Expression Conditions:

• Induction with 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Post-induction growth at lower temperatures (16-20°C for 16-18 hours) to enhance solubility

• Supplementation with 1% glucose may help reduce basal expression

Purification Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl (pH 7.5), 70 mM NH4Cl, 30 mM KCl, and protease inhibitors

• Initial purification using Ni-NTA affinity chromatography

• Further purification by ion-exchange chromatography (typically using Q-Sepharose)

• Final polishing step using size-exclusion chromatography

• Storage in buffer containing 50 mM Tris-HCl (pH 7.5), 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2, and 10% glycerol

For obtaining highly active protein, it's critical to ensure proper folding and GTP-binding capacity throughout the purification process.

How can researchers verify the functional activity of purified recombinant Marinomonas sp. IF-2?

Verification of functional activity for recombinant Marinomonas sp. IF-2 should include multiple assays targeting different aspects of IF-2 function:

GTP Binding and Hydrolysis:

• Measure GTP binding using fluorescently labeled GTP analogs (e.g., mant-GTP)

• Assess GTPase activity through phosphate release assays (e.g., malachite green assay)

30S Binding Assays:

• Use fluorescence-based approaches with labeled IF-2 variants (such as IF2(Atto)) to measure binding kinetics to 30S subunits

• Determine association (kon) and dissociation (koff) rate constants, which should yield a Kd value in the range of 40 nM for IF-2·GTP binding to 30S subunits

fMet-tRNA Interaction:

• Assess fMet-tRNA binding using filter binding assays with 35S-labeled fMet-tRNA

• Measure FRET between fluorescein-labeled fMet-tRNA and the IF-2 protein to determine interaction kinetics

• Verify that the Kd for the IF-2·GTP·fMet-tRNA complex is approximately 1 μM

30S Initiation Complex Formation:

• Monitor the ability of IF-2 to accelerate 30S initiation complex formation using FRET between labeled components

• Confirm that IF-2 promotes binding of fMet-tRNA to the 30S subunit with an association rate constant of approximately 5 μM-1s-1

50S Joining Assay:

• Measure the rate of 50S subunit joining to the 30S initiation complex using light scattering techniques

• Verify that IF-2 facilitates this process as expected for functional bacterial IF-2

A functional IF-2 should demonstrate activity in all these assays, with kinetic parameters similar to those reported for other bacterial IF-2 proteins.

What methodologies can be used to study the interaction of Marinomonas sp. IF-2 with the ribosome?

Several complementary approaches can be employed to study the interaction of Marinomonas sp. IF-2 with the ribosome:

Chemical Probing:

• Base-specific chemical probing can identify ribosomal RNA regions protected by IF-2 binding

• Focus on domains V and VI of 23S rRNA, particularly positions A2476, A2478, and the sarcin-ricin loop around position 2660, which are known IF-2 binding sites

• DMS (dimethyl sulfate) can be used to probe adenine residues, while CMCT can be used for uracil residues

Fluorescence-Based Approaches:

• FRET experiments using fluorescently labeled IF-2 (such as IF2(Atto)) and ribosomal components (like IF3(Alx))

• Stopped-flow kinetic measurements to determine binding rates and affinities

• Fluorescence anisotropy to measure direct binding interactions

Cryo-Electron Microscopy:

• Structure determination of Marinomonas sp. IF-2 bound to the 30S or 70S ribosome

• Analysis of conformational changes induced by IF-2 binding

• Comparison with existing structures from other bacterial species

Cross-linking Studies:

• Site-specific cross-linking between engineered cysteines in IF-2 and ribosomal components

• Mass spectrometry analysis to identify cross-linked residues

• Mapping of interaction interfaces

Ribosome Binding Assays:

• Sucrose density gradient analysis to assess the effect of IF-2 on subunit association

• Filter binding assays to quantify the binding of IF-2 to ribosomes under various conditions

• Competition assays with known ribosome-binding antibiotics or factors

A combination of these approaches provides a comprehensive understanding of how Marinomonas sp. IF-2 interacts with the ribosome, potentially revealing species-specific adaptations in this marine bacterium.

What are the key structural domains of Marinomonas sp. IF-2 and how do they contribute to its function?

Bacterial IF-2, including that from Marinomonas species, typically consists of several functional domains that work together to facilitate translation initiation:

N-terminal Domain:

• Highly variable in length and sequence among bacterial species

• Functions primarily in interactions with the ribosome

• May contain species-specific adaptations in Marinomonas related to its marine environment

G-Domain (Domain I):

• Contains the GTP-binding and hydrolysis machinery

• Highly conserved among GTPases

• Undergoes conformational changes upon GTP binding and hydrolysis that regulate IF-2 function

• Critical for promoting 50S subunit joining to the 30S initiation complex

Domain II:

• Contributes to ribosome binding

• May interact with the sarcin-ricin loop of 23S rRNA (positions around 2660)

• Moderately conserved among bacterial species

Domain III:

• Contains the C-terminal part of the protein

• Specifically recognizes and interacts with the initiator tRNA (fMet-tRNA)

• Helps position the initiator tRNA correctly in the P-site of the ribosome

GTP-Binding Pocket:

• Located within the G-domain

• Contains conserved sequence motifs (G1-G5) found in all GTPases

• Coordination of Mg2+ ion essential for GTP hydrolysis

The interfaces between these domains undergo significant conformational rearrangements during the translation initiation process, particularly upon GTP hydrolysis, which is triggered by 50S subunit joining. These conformational changes likely facilitate the release of IF-2 from the 70S initiation complex, allowing translation to proceed to the elongation phase .

How does the ribosome binding of Marinomonas sp. IF-2 compare to that of other bacterial species?

Based on the available research data, the ribosome binding of IF-2 appears to be conserved across bacterial species, suggesting Marinomonas sp. IF-2 likely follows similar patterns:

Binding Sites on the Ribosome:

• IF-2 specifically protects nucleotides A2476 and A2478 in domain V of 23S rRNA

• It also protects residues around position 2660 in domain VI (the sarcin-ricin loop)

• These protection patterns appear to be universal features of bacterial IF-2

Binding Independence:

• IF-2 binding to the 30S subunit occurs independently of fMet-tRNA, GTP, mRNA, and IF1

• This binding characteristic appears to be conserved across bacterial species

Effect on Subunit Association:

• IF-2 has a "tightening effect" on ribosomal subunit association, as indicated by decreased reactivity of residues A1418 and A1483 in 16S rRNA

• This property seems to be universally conserved across bacterial species

Binding Kinetics:

• The association rate constant for IF-2·GTP binding to the 30S subunit is approximately 25 μM-1s-1

• The dissociation rate constant is approximately 1 s-1

• These result in a Kd value of about 40 nM

While the fundamental mechanism of ribosome binding appears conserved, species-specific adaptations in Marinomonas sp. IF-2 may exist, particularly in the N-terminal region which is the most variable part of the protein among bacterial species. These adaptations could be related to the marine environment where Marinomonas species typically live, potentially affecting the strength or specificity of ribosome interactions under conditions of varying salinity or temperature.

What role does GTP hydrolysis play in the function of Marinomonas sp. IF-2?

GTP hydrolysis is a critical regulatory mechanism in the function of bacterial IF-2, including Marinomonas sp. IF-2. The process follows several distinct steps with specific functional consequences:

GTP Binding:

• IF-2 binds GTP with high affinity prior to interacting with the 30S ribosomal subunit

• GTP-bound IF-2 (IF-2·GTP) is the active form that efficiently promotes initiator tRNA binding

• The GTP-bound state stabilizes a conformation of IF-2 that optimally interacts with both the ribosome and fMet-tRNA

Timing of GTP Hydrolysis:

• GTP hydrolysis is triggered upon 50S subunit joining to the 30S initiation complex

• This timing ensures that GTP hydrolysis occurs only after proper assembly of the initiation complex

• The energy from GTP hydrolysis is not used for fMet-tRNA binding or delivery (unlike with EF-Tu)

Functional Consequences:

• GTP hydrolysis induces conformational changes in IF-2 that reduce its affinity for the ribosome

• These changes facilitate the release of IF-2 from the 70S initiation complex

• Release of IF-2 allows the ribosome to proceed to the elongation phase of translation

• GTP hydrolysis also serves as a proofreading mechanism, ensuring that only correctly formed initiation complexes proceed to elongation

Regulation:

• The GTPase activity of IF-2 is intrinsically low but is stimulated by interaction with the ribosome

• Specifically, the GTPase-activating center of the ribosome (likely involving the sarcin-ricin loop) triggers hydrolysis

• This represents a critical control point in translation initiation

The role of GTP hydrolysis in IF-2 function differs fundamentally from that in elongation factors like EF-Tu, where GTP hydrolysis drives tRNA selection. In IF-2, GTP hydrolysis primarily serves as a timing mechanism to ensure ordered assembly and disassembly of the translation initiation machinery .

How does Marinomonas sp. IF-2 compare to IF-2 from model organisms like E. coli?

While specific comparative data for Marinomonas sp. IF-2 is limited in the provided search results, we can infer likely similarities and differences based on general patterns of IF-2 conservation and variation among bacterial species:

Sequence Conservation:

• The G-domain (containing GTP-binding motifs) is likely highly conserved between Marinomonas sp. and E. coli IF-2

• Domain III, which interacts with initiator tRNA, is also expected to show high conservation

• The N-terminal domain likely exhibits the greatest sequence divergence, as this is the most variable region among bacterial IF-2 proteins

Functional Conservation:

• Core functions such as GTP binding, fMet-tRNA recognition, and promotion of 50S subunit joining are likely conserved

• The mechanism of action, with IF-2·GTP binding to the 30S subunit first rather than delivering fMet-tRNA as a carrier, is probably shared between the species

• Ribosome binding sites, particularly protection of A2476, A2478, and the sarcin-ricin loop region, are likely conserved

Potential Adaptations in Marinomonas:

• Marinomonas species are typically marine bacteria that may have adapted to function optimally in conditions of higher salinity

• Possible adaptations in the N-terminal domain might affect interactions with the ribosome under marine-specific conditions

• Kinetic parameters of GTP hydrolysis and ribosome binding might be optimized for temperatures typical of marine environments

Comparative Binding Parameters:

• E. coli IF-2·GTP binds to the 30S subunit with a Kd of approximately 40 nM

• E. coli IF-2·GTP forms a complex with fMet-tRNA with a Kd of approximately 1 μM

• Similar values would be expected for Marinomonas sp. IF-2, with potentially small variations reflecting environmental adaptations

A detailed comparative analysis would require direct experimental comparison of recombinant Marinomonas sp. IF-2 with E. coli IF-2 under identical conditions, examining parameters such as thermal stability, salt tolerance, GTPase activity, and ribosome binding kinetics.

What evolutionary adaptations might be present in Marinomonas sp. IF-2 related to its marine environment?

Marinomonas species are predominantly marine bacteria, and their translation machinery, including IF-2, may have evolved specific adaptations to function optimally in marine environments:

Salt Tolerance Adaptations:

• Increased proportion of acidic amino acids on the protein surface to maintain solubility in higher salt concentrations

• Modified electrostatic interactions at protein-RNA interfaces to maintain optimal binding to the ribosome in varying salt conditions

• Potentially altered GTP binding pocket characteristics to ensure proper nucleotide binding despite ionic strength variations

Temperature Adaptations:

• If the specific Marinomonas species inhabits cold marine environments, IF-2 might show cold-adaptation features such as reduced proline content and increased flexibility of loop regions

• Alternatively, for Marinomonas species in warmer waters, increased thermostability through additional salt bridges or hydrophobic core packing

• Optimized kinetic parameters for GTP hydrolysis at the environmental temperatures typically encountered

Pressure Adaptations (for deep-sea species):

• Modified volume changes during conformational transitions to function under elevated hydrostatic pressure

• Structural modifications that reduce the sensitivity of protein-protein and protein-RNA interactions to pressure

Codon Usage Adaptation:

• The infB gene in Marinomonas might show codon usage patterns optimized for translation efficiency under marine conditions

• This adaptation would ensure adequate levels of IF-2 production despite potentially different tRNA abundances in marine bacteria

Co-evolution with Marine Ribosomal RNA:

• Fine-tuned binding interactions with potentially unique features of Marinomonas ribosomal RNA

• Adaptations in the regions that interact with the sarcin-ricin loop and other ribosomal binding sites

Regulatory Adaptations:

• Modified regulatory mechanisms for IF-2 expression in response to marine-specific environmental stresses

• Potential integration with marine-specific stress response pathways

Comparative genomic and biochemical studies between Marinomonas sp. IF-2 and homologs from terrestrial bacteria would be valuable to identify and characterize these potential adaptations. Such research could provide insights into molecular mechanisms of environmental adaptation in translation machinery.

How does the function of bacterial IF-2 compare to its eukaryotic counterparts?

Bacterial IF-2, including that from Marinomonas species, differs significantly from its eukaryotic counterparts in structure, function, and mechanism:

Comparison with eIF2 (eukaryotic initiation factor 2):

• Function: eIF2 forms a ternary complex with GTP and Met-tRNAi and delivers it to the 40S ribosomal subunit, functioning as a true tRNA carrier. In contrast, bacterial IF-2 binds to the 30S subunit first and then promotes fMet-tRNA binding

• Structure: eIF2 is a heterotrimeric protein (α, β, and γ subunits), whereas bacterial IF-2 is a single polypeptide

• Regulation: eIF2 is subject to extensive regulation through phosphorylation of its α subunit in response to cellular stress, a regulatory mechanism absent in bacterial IF-2

• Binding Affinity: eIF2 forms much tighter complexes with initiator tRNA (Kd in nM range) compared to the relatively weak binding of bacterial IF-2 (Kd ≈ 1 μM)

Comparison with eIF5B (eukaryotic initiation factor 5B):

• Functional Similarity: eIF5B is the true functional homolog of bacterial IF-2, primarily promoting subunit joining rather than acting as a tRNA carrier

• Structural Homology: eIF5B shares structural homology with bacterial IF-2, particularly in the G-domain and domain II

• tRNA Interaction: Like bacterial IF-2, eIF5B has low affinity for initiator tRNA (Kd > 5 μM or 40 μM according to different studies)

• Evolution: The common activity of eIF5B and bacterial IF-2 in accelerating subunit joining was conserved throughout evolution, while the ability to protect initiator tRNA was lost by eIF5B, as this function was taken over by eIF2 in eukaryotes

Mechanistic Differences in Translation Initiation:

• Bacterial initiation involves three initiation factors (IF1, IF2, IF3), while eukaryotic initiation involves at least twelve factors (eIF1 through eIF6)

• Bacterial initiation directly positions the mRNA on the 30S subunit via interaction between the Shine-Dalgarno sequence and 16S rRNA, whereas eukaryotic initiation involves a scanning mechanism to locate the start codon

• The energy from GTP hydrolysis serves different functions: in bacteria, IF-2-catalyzed GTP hydrolysis primarily drives factor release, while in eukaryotes, multiple GTP hydrolysis events regulate various steps of the initiation process

These differences highlight the divergent evolution of translation initiation mechanisms between bacteria and eukaryotes, with bacterial systems generally being simpler and more direct.

How can site-directed mutagenesis of Marinomonas sp. IF-2 be used to investigate its mechanism of action?

Site-directed mutagenesis represents a powerful approach for investigating the structure-function relationships in Marinomonas sp. IF-2. Key experimental strategies and target regions include:

GTP-Binding Pocket Mutations:

• G-Domain Motifs: Mutations in conserved GTP-binding motifs (G1-G5) can help elucidate the role of GTP binding and hydrolysis

• Catalytic Residues: Substitution of residues involved in GTP hydrolysis (e.g., histidine residues that position water for nucleophilic attack) can create GTPase-deficient variants

• Expected Outcomes: Such mutants would likely bind GTP normally but fail to hydrolyze it, remaining locked in the GTP-bound state and potentially inhibiting ribosome recycling

fMet-tRNA Binding Domain Mutations:

• C-Terminal Domain: Targeted mutations in domain III, which interacts with the 3' end of fMet-tRNA

• Interface Residues: Focus on conserved residues at the IF-2/fMet-tRNA interface

• Expected Outcomes: Reduced affinity for fMet-tRNA, leading to defects in 30S initiation complex formation

Ribosome Binding Interface Mutations:

• N-Terminal Region: Mutations in the variable N-terminal domain to assess its role in ribosome binding

• Domain II Residues: Alterations in residues that interact with the sarcin-ricin loop of 23S rRNA

• Expected Outcomes: Altered kinetics of ribosome binding or reduced protection of specific rRNA nucleotides in chemical probing experiments

Interdomain Communication Mutations:

• Hinge Regions: Mutations at interdomain boundaries to investigate conformational changes during the initiation cycle

• Allosteric Sites: Identification and mutation of residues involved in transmitting conformational changes between domains

• Expected Outcomes: Disruption of the coordination between GTP hydrolysis and factor release from the ribosome

Experimental Approaches with Mutants:

This systematic mutagenesis approach would provide valuable insights into the mechanism of action of Marinomonas sp. IF-2 and potentially reveal species-specific adaptations relevant to its function in marine environments.

What experimental approaches can be used to investigate the role of Marinomonas sp. IF-2 in stress response?

Investigating the role of Marinomonas sp. IF-2 in stress response requires a multifaceted approach combining genetics, biochemistry, and systems biology techniques:

Transcriptional and Translational Regulation Studies:

Genetic Manipulation Approaches:

• Generation of conditional infB mutants (since complete deletion would likely be lethal)

• Construction of strains with modified infB promoters to alter expression levels

• Introduction of heterologous IF-2 proteins from non-marine bacteria to assess functional complementation

• CRISPR interference (CRISPRi) to achieve tunable repression of infB expression

Biochemical Characterization Under Stress Conditions:

• Analysis of GTPase activity of purified IF-2 under varying salt concentrations, temperatures, and pH

• Assessment of fMet-tRNA binding efficiency under stress conditions

• Determination of ribosome binding kinetics at different temperatures and salt concentrations

• Investigation of potential stress-induced post-translational modifications of IF-2

In vivo Translation Dynamics:

• Pulse-chase experiments with radiolabeled amino acids to assess translation rates under stress

• Polysome profiling to examine ribosome distribution during stress response

• Use of reporter constructs to monitor translation initiation efficiency under various stresses

• Single-cell analysis of translation using fluorescent reporters to capture cell-to-cell variability

Systems Biology Approaches:

• Integration of transcriptomics, proteomics, and metabolomics data to place IF-2 function within the broader stress response network

• Network analysis to identify stress-specific interaction partners of IF-2

• Mathematical modeling of translation initiation under stress conditions

The marine environment presents unique stresses including salinity fluctuations, pressure changes, and temperature variations. Understanding how Marinomonas sp. IF-2 functions under these conditions may reveal novel adaptations in translation initiation machinery that could have broader implications for understanding bacterial adaptation to extreme environments.

How can cryo-electron microscopy be optimized for studying Marinomonas sp. IF-2 bound to the ribosome?

Cryo-electron microscopy (cryo-EM) has revolutionized our understanding of ribosome complexes, but requires careful optimization for studying specific factors like Marinomonas sp. IF-2. Here are key considerations and strategies:

Sample Preparation Optimization:

Data Collection Strategies:

• Use of energy filters to improve signal-to-noise ratio, particularly important for visualizing dynamic factors like IF-2

• Collection of tilt pairs or tilt series to overcome preferred orientation issues

• Implementation of beam-tilt data collection for aberration correction

• Use of movies with dose fractionation to correct for beam-induced motion

• Employment of phase plates to enhance contrast of small features

Computational Analysis Approaches:

• Application of 3D classification to separate different conformational states of IF-2

• Focused refinement on the IF-2 region to improve local resolution

• Use of multi-body refinement to account for domain movements within IF-2

• Implementation of time-resolved cryo-EM by vitrifying samples at different time points after GTP addition

• Integration with molecular dynamics simulations to model conformational changes

Validation and Complementary Techniques:

• Cross-validation with chemical probing data, particularly for identified RNA contacts

• Comparison with directed hydroxyl radical probing to confirm protein-RNA interfaces

• Integration with mass spectrometry data from cross-linking experiments

• Correlation with functional data from mutagenesis studies

• Benchmarking against existing structures from model organisms

Expected Structural Insights:

• Visualization of specific contacts between Marinomonas sp. IF-2 and the sarcin-ricin loop

• Identification of potential marine-specific adaptations in the IF-2 structure

• Characterization of conformational changes between different states of the initiation complex

• Detailed understanding of how IF-2 positions fMet-tRNA for optimal interaction with the start codon

By optimizing these parameters, researchers can obtain high-resolution structures of Marinomonas sp. IF-2 on the ribosome, potentially revealing unique adaptations of the translation machinery in this marine bacterium.